Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Micro BCA TM Protein Assay Kit , ThermoFisher Scientific , Cat# #23235 RRID: N/A.

Techniques: Control, Affinity Purification, Recombinant, Bicinchoninic Acid Protein Assay, Western Blot, Electron Microscopy, RNAscope, Multiplex Assay, Mass Spectrometry, Mutagenesis, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding

doi: 10.1074/jbc.RA118.006159

Figure Lengend Snippet: NSD2 interacting partners identified by BioID. A, study design: TKO control cells or TKO MM cells stably expressing NSD2-BirA were cultured in media supplemented with 50 μm biotin for 48 h and then lysed. Biotinylated proteins were captured by streptavidin beads. On-bead trypsin digestion was performed and the resulting peptides were desalted and identified by nanocapillary LC-MS. B, immunoblot of TKO cells stably expressing empty vector, NSD2, or NSD2-BirA. Because the NSD2-BirA construct contains a C-terminal HA tag, the HA tag blot confirmed expression of the NSD2-BirA construct in TKO cells. C, a Venn diagram depicting unique and common interacting proteins identified in NSD2-BirA cells and TKO control cells. D, gene set enrichment analysis of NSD2-binding partners organized according to GO biological process using EnrichR (28).

Article Snippet: Pierce TM Streptavidin Magnetic Beads (60 μl, Thermo Fisher Scientific) were washed 3 times with lysis buffer and incubated with protein lysate overnight at 4 °C on a tube rotator.

Techniques: Control, Stable Transfection, Expressing, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Western Blot, Plasmid Preparation, Construct, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding

doi: 10.1074/jbc.RA118.006159

Figure Lengend Snippet: NSD2-binding partners identified by label-free quantitative MS. A, study design: three biological replicates of TKO cells stably expressing NSD2-BirA or nuclear-localized BirA control were cultured with biotin, lysed, and quantified. Equal amounts of total protein from each sample was used for streptavidin capture and trypsin digestion. The resulting peptides were purified and analyzed by nanocapillary LC-MS in triplicate. High resolution MS1 was used for relative peptide quantification and MS2 was used for protein identification. B, volcano plot of quantified proteins is shown. The x axis is the fold-change in protein abundance of NSD2-BirA relative to BirA control. The y axis (instantaneous q-value) measures the statistical confidence of the variation between NSD2-BirA and BirA control. The vertical dotted lines represent a 2-fold change threshold. The horizontal dotted line indicates a false discovery rate of 5%. Twenty-four nuclear targets above threshold are colored red. C, box and whisker plot indicating variations in signal intensity of each quantified protein. Variation attributed to the treatment (NSD2-BirA versus BirA control), biological replicates, technical replicates, or residuals (variation not explained by any of the previously described sources) are indicated. D, gene ontology KEGG pathway analysis of the 24 high confidence NSD2 partners. E, Venn diagram of overlapping hits from the BioID IP-MS qualitative Study 1 (see Fig. 1) and label-free quantitative Study 2.

Article Snippet: Pierce TM Streptavidin Magnetic Beads (60 μl, Thermo Fisher Scientific) were washed 3 times with lysis buffer and incubated with protein lysate overnight at 4 °C on a tube rotator.

Techniques: Binding Assay, Stable Transfection, Expressing, Control, Cell Culture, Purification, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Whisker Assay, Protein-Protein interactions

Journal: The Journal of Biological Chemistry

Article Title: Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding

doi: 10.1074/jbc.RA118.006159

Figure Lengend Snippet: PARylation of NSD2 disrupts its association with chromatin in vitro. A, schematic of the experimental strategy. After the PARylation reaction, SAM and biotinylated HeLa oligonucleosomes were allowed to bind to NSD2. Streptavidin magnetic beads were added into the reaction and incubated for 1 h at room temperature. B, biotinylated nucleosomes along with bound NSD2 were captured and analyzed by immunoblot against NSD2, histone H3K36me2, and total histone H4. C, histone H3K36me2 band relative intensities were quantified using ImageJ. Two independent experiments are shown.

Article Snippet: Pierce TM Streptavidin Magnetic Beads (60 μl, Thermo Fisher Scientific) were washed 3 times with lysis buffer and incubated with protein lysate overnight at 4 °C on a tube rotator.

Techniques: In Vitro, Magnetic Beads, Incubation, Western Blot